PROJECT SUMMARY ?-Lactams are drugs of vital importance for treatment of bacterial infections. However, the emergence of bacterial metallo-?-lactamase (MBL) enzymes has enabled resistance to almost all clinically used drugs in this class (excepting the monobactams). Arguably, the most worrisome MBL to emerge is New Dehli Metallo-?- lactamase-1 (NDM-1), which can hydrolyze all generations of bicyclic ?-lactams that have been tested, including penems, cephems, and carbapenems. NDM-1 has been identified in a number of different Enterobacteriaceae, and is community-acquired (not just nosocomial). The gene is encoded in plasmids capable of horizontal transfer that also confer resistance to macrolides, aminoglycosides, rifampicin, sulfamethoxazole, and monobactams. Therefore MBLs, and NDM-1 in particular, represent a significant emerging worldwide health threat. Despite its importance, drugs that target NDM-1, or any other MBL, are not known. At the time of writing, there are only ~11, relatively weak inhibitors reported for NDM-1, and inhibitors of the other most clinically-relevant MBLs are limited in efficacy and chemical diversity. Inhibitor development has been difficult, in part, due to the flexible active sites of these enzymes and the challenges associated with targeting metalloenzymes. This proposal will directly address this deficit by using innovative fragment-based drug discovery complimented with high-throughput screening approaches, detailed characterization of inhibitor binding, bioinorganic spectroscopy, kinetics, X-ray crystallography, and assays of microbial efficacy. We will conduct a deep investigation and optimization of two conserved features of MBL inhibitors: metal-binding groups and substituents that interact with the flexible loops of the protein. Aim 1 uses privileged chelator- fragment libraries to identify moieties effective for inhibition of NDM-1 and other clinically-relevant B1 lactamases. Aim 2 uses diversity screening to identify and optimize structurally divergent inhibitors to elucidate how the flexible NDM-1 binding sites accommodate different ligands. Aim 3 uses a battery of structural and spectroscopic techniques to characterize, re-design, and ultimately optimize these inhibitors and their interactions with NDM-1. An experienced team brings proficiency in organic synthesis, inhibitor screening, medicinal chemistry, enzymology, spectroscopy, structural biology, and microbiology to advance hits into potent NDM-1 inhibitors with known mechanisms of action and microbial efficacy. This work will make critical contributions to validating novel, potent, structurally-diverse, and biologically-useful NDM-1 inhibitors. The proposed work is innovative due to the application of a unique chelator-fragment discovery approach, the extensive inhibitor-target characterization relevant to many B1 lactamases, and the multidisciplinary team committed to developing much needed first-in-class inhibitors as tools and lead compounds for therapeutic design to counter the global threat of NDM-1.